S.J. Watson

Altered cortical glutamatergic and GABAergic signal transmission with glial involvement in depression

Abnormalities in l-glutamic acid (glutamate) and GABA signal transmission have been postulated to play a role in depression, but little is known about the underlying molecular determinants and neural mechanisms. Microarray analysis of specific areas of cerebral cortex from individuals who had suffered from major depressive disorder demonstrated significant down-regulation of SLC1A2 and SLC1A3, two key members of the glutamate/neutral amino acid transporter protein family, SLC1. Similarly, expression of l-glutamate-ammonia ligase, the enzyme that converts glutamate to nontoxic glutamine was significantly decreased. Together, these changes could elevate levels of extracellular glutamate considerably, which is potentially neurotoxic and can affect the efficiency of glutamate signaling. The astroglial distribution of the two glutamate transporters and l-glutamate-ammonia ligase strongly links glia to the pathophysiology of depression and challenges the conventional notion that depression is solely a neuronal disorder. The same cortical areas displayed concomitant up-regulation of several glutamate and GABAA receptor subunits, of which GABAAα1 and GABAAβ3 showed selectivity for individuals who had died by suicide, indicating their potential utility as biomarkers of suicidality. These findings point to previously undiscovered molecular underpinnings of the pathophysiology of major depression and offer potentially new pharmacological targets for treating depression.

Gene expression of prohormone and proprotein convertases in the rat CNS: a comparative in situ hybridization analysis

Posttranslational processing of proproteins and prohormones is an essential step in the formation of bioactive peptides, which is of particular importance in the nervous system. Following a long search for the enzymes responsible for protein precursor cleavage, a family of Kexin/subtilisin-like convertases known as PC1, PC2, and furin have recently been characterized in mammalian species. Their presence in endocrine and neuroendocrine tissues has been demonstrated. This study examines the mRNA distribution of these convertases in the rat CNS and compares their expression with the previously characterized processing enzymes carboxypeptidase E (CPE) and peptidylglycine alpha-amidating monooxygenase (PAM) using in situ hybridization histochemistry. Furin mRNA was ubiquitously distributed and detected both in neurons and non- neuronal tissue throughout the brain with a higher abundance in ependyma, the circumventricular organs, the islands of Calleja, hippocampus, and allocortex. The cellular localization of PC1 and PC2 was exclusively neuronal with highest concentrations in known neuropeptide-rich brain regions. In general, PC2 was more widely expressed than PC1 in the CNS, although many regional variations were detected. The identification of specific combinations of convertase expression together with CPE and PAM expression in neuropeptide-rich brain regions suggests that specific enzymatic pathways are involved in neuropeptide precursor processing, and that these specific combinations are responsible for region-specific differences of posttranslational processing.

Cellular localization and distribution of the cloned mu and kappa opioid receptors in rat gastrointestinal tract.

Several pharmacological and electrophysiological studies have shown that the opioid receptors are widely distributed in the gastrointestinal tract. Despite such consensus, there are conflicting findings regarding their effects in intestinal function, and their precise site of action remained unclear. The aim of the present study was therefore to delineate the cellular localization of mu and kappa opioid receptors in rat gastrointestinal tract using polyclonal antibodies generated to C-terminal end of the cloned mu (63 amino acids) and kappa (41 amino acids) receptors. The distribution of mu differs from that of kappa receptors within the gastrointestinal wall, with a greater abundance of mu receptor-like immunoreactive fibres in all intestinal layers. Numerous neurons expressing mu receptor-like proteins were found in the submucosal plexus with comparatively few in the myenteric plexus. In contrast, a higher number of neurons expressing kappa receptor-like immunoreactivity were visualized in the myenteric plexus with a small number in the submucosal plexus. A high number of immunopositive neurons were found in the myenteric plexus of the stomach and the proximal colon with both antibodies. In the submucosal and mucosal layers. mu receptor-immunoreactive fibres were more abundant and distributed around the crypts, blood vessels and lymphatic nodes. Interestingly, numerous mu and fewer kappa receptor-immunoreactive interstitial cells are localized in the region of myenteric plexus and at the internal border of the circular muscle. Finally, smooth muscle cells did not demonstrate any mu- nor kappa-receptor immunoreactivity. These findings suggest that in the rat gastrointestinal tract, mu and kappa opioid receptors may directly influence neuronal and interstitial cell activity. This appears not to be the case for the smooth muscle cells. In the muscular layers, the anatomical data point to mu receptor actions being mediated by nerve terminals, whereas kappa receptor effects may be mediated by both nerve terminals and somatodendritic synaptic mechanisms. In contrast, in the submucosal and mucosal layers, mu receptors predominate and are localized on both nerve terminals and somatodendritic synaptic elements.

Mitochondrial-related gene expression changes are sensitive to agonal-pH state: implications for brain disorders

Mitochondrial defects in gene expression have been implicated in the pathophysiology of bipolar disorder and schizophrenia. We have now contrasted control brains with low pH versus high pH and showed that 28% of genes in mitochondrial-related pathways meet criteria for differential expression. A majority of genes in the mitochondrial, chaperone and proteasome pathways of nuclear DNA-encoded gene expression were decreased with decreased brain pH, whereas a majority of genes in the apoptotic and reactive oxygen stress pathways showed an increased gene expression with a decreased brain pH. There was a significant increase in mitochondrial DNA copy number and mitochondrial DNA gene expression with increased agonal duration. To minimize effects of agonal-pH state on mood disorder comparisons, two classic approaches were used, removing all subjects with low pH and agonal factors from analysis, or grouping low and high pH as a separate variable. Three groups of potential candidate genes emerged that may be mood disorder related: (a) genes that showed no sensitivity to pH but were differentially expressed in bipolar disorder or major depressive disorder; (b) genes that were altered by agonal-pH in one direction but altered in mood disorder in the opposite direction to agonal-pH and (c) genes with agonal-pH sensitivity that displayed the same direction of changes in mood disorder. Genes from these categories such as NR4A1 and HSPA2 were confirmed with Q-PCR. The interpretation of postmortem brain studies involving broad mitochondrial gene expression and related pathway alterations must be monitored against the strong effect of agonal-pH state. Genes with the least sensitivity to agonal-pH could present a starting point for candidate gene search in neuropsychiatric disorders.

Opiate binding properties of naturally occurring N- and C-Terminus modified beta-endorphins

Beta-endorphin is further processed within the pituitary and brain by either N-terminal acetylation, carboxy-terminal proteolysis, or both. These naturally occurring analogues are stored intracellularly and, in some tissues, represent the majority of beta-endorphin immunoreactivity detected by antisera. It is therefore critical to determine their relative potencies at the opiate receptor. This study demonstrates that cleavage of the C-terminus tetrapeptide brings about a 10-fold decrease in opiate binding potency of either camel or human beta-endorphin. N-Acetylation, on the other hand, causes over a thousand fold loss in opiate potency rendering the peptide effectively inactive. Since unmodified beta-endorphin is approximately equipotent at multiple opiate receptors, we tested for possible differential shifts towards mu or delta-type receptors which may result from the modification. Our results show no change in selectivity, but simply an overall loss of potency.

Immunohistochemical localization of the cloned κ1 receptor in the rat CNS and pituitary

Abstract Several lines of evidence have demonstrated the presence of three opioid receptor types in the CNS and periphery. These receptors are referred to as μ, δ and κ, and have been implicated in a wide variety of functions. The present study examines the localization of the κ 1 receptor-like protein using antibodies generated to the C terminal 42 amino acids of the cloned κ 1 receptor, a region of the receptor that has little homology with μ and b receptors. Immunohistochemical studies in Zamboni-fixed rat tissue demonstrate immunoreactive perikarya and/or fibers in such regions as the deep layers of the parietal, temporal and occipital cortex, parasubiculum, central and medial amygdala, bed nucleus stria terminalis, nucleus accumbens, olfactory tubercle, endopiriform nucleus, claustrum, hypothalamic nuclei, median eminence, midline thalamic nuclei, zona incerta, central gray, caudal linear and dorsal raphe, substantia nigra, pars reticulata, ventral tegmental area, parabrachial nucleus, spinal trigeminal nucleus, nucleus of the solitary tract, spinal cord and the dorsal root ganglia. Specific κ 1 receptor-like immunohistochemical staining is also observed in the pituitary, where immunoreactive perikarya and fibers are localized in the neural and intermediate lobes. Transfection and preabsorption controls suggest that the antibody is selective for the cloned κ 1 receptor, and does not recognize μ or δ. This immunohistochemical localization corresponds well to previously described κ 1 receptor mRNA and binding distributions and provides new insights into the cellular localization and pre- and postsynaptic organization of the κ 1 receptor-like proteins in the rat brain and pituitary. The functional implications of these results are discussed in light of the role K, receptors play in hormonal regulation, antinociception and reward.

Nociceptin/orphanin FQ regulates neuroendocrine function of the limbic-hypothalamic-pituitary-adrenal axis

We examined the effects of the neuropeptide nociceptin/orphanin FQ on activity of the limbic-hypothalamic-pituitary-adrenal axis (also known as the stress axis) in rats. This axis regulates important metabolic functions, and initiates critical neuroendocrine responses that cope with environmental threats and challenges to homeostatic functioning. Disregulation of the limbic-hypothalamic-pituitary-adrenal axis is associated with impaired physical and psychological health. In the present experiments, rats were treated with intracerebroventricular injections of nociceptin/orphanin FQ in the presence or absence of acute stressors. Plasma adrenocorticotrophic hormone and corticosterone concentrations were assayed 15 or 30min after injections. In the rats that were not exposed to stress, nociceptin/orphanin FQ produced dose-orderly elevations of circulating adrenocorticotrophic hormone and corticosterone concentrations. These effects were also found after administration of the nociceptin/orphanin FQ analogues, des-Phe orphanin FQ and [Phe(1)psi(CH(2)-NH)Gly(2)]nociceptin((1-13))NH(2). In rats that were exposed to the mild stress of a novel environment, nociceptin/orphanin FQ administration enhanced the stress-induced elevations of plasma adrenocorticotrophic hormone concentrations and prolonged the stress-induced elevations of plasma corticosterone concentrations. In rats that were exposed to restraint stress, nociceptin/orphanin FQ administration did not augment the stress-induced elevations in plasma hormones, perhaps because of a ceiling effect. We conclude that administration of nociceptin/orphanin FQ activates neuroendocrine activity of the limbic-hypothalamic-pituitary-adrenal axis even in the absence of a stressor, and may delay the shutdown of these physiological responses after exposure to acute mild stress. In light of the known functions of this axis, it appears that nociceptin/orphanin FQ participates in the regulation of important metabolic functions, and may be implicated in physiological responses to stress. This interaction between nociceptin/orphanin FQ and the limbic-hypothalamic-pituitary-adrenal axis implicates nociceptin/orphanin FQ in important aspects of physiological and psychological well-being.

Relation between the Hypothalamic-Pituitary-Thyroid (HPT) Axis and the Hypothalamic-Pituitary-Adrenal (HPA) Axis during Repeated Stress

Previous work has indicated that acute and repeated stress can alter thyroid hormone secretion. Corticosterone, the end product of hypothalamic-pituitary-adrenal (HPA) axis activation and strongly regulated by stress, has been suggested to play a role in hypothalamic-pituitary-thyroid (HPT) axis regulation. In the current study, we sought to further characterize HPT axis activity after repeated exposure to inescapable foot-shock stress (FS), and to examine changes in proposed regulators of the HPT axis, including plasma corticosterone and hypothalamic arcuate nucleus agouti-related protein (AGRP) mRNA levels. Adult male Sprague-Dawley rats were subjected to one daily session of inescapable FS for 14 days. Plasma corticosterone levels were determined during and after the stress on days 1 and 14. Animals were killed on day 15, and trunk blood and brains were collected for measurement of hormone and mRNA levels. Repeated exposure to FS led to a significant decrease in serum levels of 3,5,3′-triiodothyronine (T3) and 3,5,3′,5′-tetraiodothyronine (T4). Stress-induced plasma corticosterone levels were not altered by repeated exposure to the stress. Despite the decrease in peripheral hormone levels, thyrotropin-releasing hormone (TRH) mRNA levels within the paraventricular nucleus of the hypothalamus were not altered by the stress paradigm. Arcuate nucleus AGRP mRNA levels were significantly increased in the animals exposed to repeated FS. Additionally, we noted significant correlations between stress-induced plasma corticosterone levels and components of the HPT axis, including TRH mRNA levels and free T4 levels. Additionally, there was a significant correlation between AGRP mRNA levels and total T3 levels. Changes in body weight were also correlated with peripheral corticosterone and TRH mRNA levels. These results suggest that repeated exposure to mild-electric foot-shock causes a decrease in peripheral thyroid hormone levels, and that components of the HPA axis and hypothalamic AGRP may be involved in stress regulation of the HPT.

Estrogen receptor α and β mRNA expressions by proliferating and differentiating cells in the adult rat dentate gyrus and subventricular zone

Numerous factors modulate neurogenesis in the adult dentate gyrus and subventricular zone, but it is often not clear if the modulation is mediated by direct effects on the proliferating and differentiating cells or secondary to effects on other cells. Also, while some factors selectively affect neurogenesis in one of the neurogenetic zones, it is not clear how selectivity is achieved. Estrogen is a hormonal modulator of neurogenesis. To address the issues of direct versus indirect control and regional specificity we investigated the colocalization of immunoreactivity for a proliferating cell marker, Ki-67, and a marker for migrating and differentiating cells with a neuronal phenotype, doublecortin, with the expressions of mRNA for estrogen receptors alpha and beta. We found an extensive colocalization of estrogen receptor alpha with both markers in the dentate gyrus and only with Ki-67 in the subventricular zone. An extensive colocalization of estrogen receptor beta with both markers was found in the dentate gyrus, but only a few Ki-67-immunoreactive and no doublecortin-immunoreactive cells of the subventricular zone expressed estrogen receptor beta mRNA. Estrogen receptor alpha and beta mRNAs were not expressed in other telencephalic Ki-67-immunoreactive cells or in constitutively doublecortin-immunoreactive cells of the piriform cortex. The extensive colocalization of immunoreactive markers for cell proliferation and differentiation with mRNAs for estrogen receptor alpha and estrogen receptor beta points to the direct modulation of dentate cell proliferation, differentiation and survival by estrogen, while direct effects of estrogen in the subventricular zone appear restricted to estrogen receptor alpha-mediated effects operating at the time of cell proliferation.

Environmental context and drug history modulate amphetamine-induced c-fos mRNA expression in the basal ganglia, central extended amygdala, and associated limbic forebrain.

The context in which amphetamine is administered modulates its ability to induce both behavioral sensitization and immediate early gene expression. When given in a novel test environment amphetamine produces greater levels of c-fos and arc mRNA expression in many brain regions relative to when it is given in the home cage. The purpose of the current study was to determine if environment and drug history interact to influence amphetamine-induced c-fos mRNA expression. Rats with a unilateral 6-hydroxydopamine lesion were treated for 7 days with saline or 0.5 mg/kg of d-amphetamine (i.v.) in a distinct and relatively novel test environment (Novel), or in their home cage (Home). Following a 10-12-day withdrawal period, a challenge injection of either saline or 0.5 mg/kg d-amphetamine was administered. In situ hybridization histochemistry was used to examine c-fos mRNA expression in several regions of the basal ganglia, the central extended amygdala, and limbic forebrain. In most brain regions amphetamine given in the Novel environment produced greater c-fos mRNA expression than when given it was given at Home, and drug history had no effect on amphetamine-induced c-fos mRNA expression. However, within the subthalamic nucleus, substantia nigra reticulata, and central nucleus of the amygdala prior experience with amphetamine in the Novel but not Home environment enhanced the effect of an amphetamine challenge injection on c-fos mRNA expression. In contrast, there was a decrease in c-fos mRNA expression in amphetamine-pretreated animals, regardless of environmental context, in the ventral portion of the far caudal striatum. Reexposure to an environment previously paired with amphetamine produced a conditioned increase in c-fos mRNA expression in portions of the caudate-putamen, the subthalamic nucleus, the nucleus accumbens shell and a conditioned decrease in c-fos mRNA expression in the central nucleus of the amygdala. We conclude that environmental context and drug history interact to alter the basal ganglia and central extended amygdala circuitry engaged by subsequent exposure to amphetamine, or exposure to an environment previously paired with amphetamine.